Many nucleic acid analogs of DNA and RNA have been synthesized and shown to have markedly different molecular recognition properties. The central feature of molecular recognition by well-ordered, intermolecular hydrogen-bonding between linear strands of nucleic acids (Blackburn, G. M. and Gait, M. J. Eds. "DNA and RNA structure" in Nucleic Acids in Chemistry and Biology, 2.sup.nd Edition, (1996) Oxford University Press, p. 15-22), can be grossly affected by structural modifications. Some analogs have greater affinity for their complementary DNA and RNA, exemplified by higher thermal melting values, T.sub.m. In this effect, affinity is synonymous with hybridization strength and duplex stability. Ideally, nucleic acid analogs demonstrate a high degree of base-discrimination following the normal Watson/Crick rules (A+T, G+C). The level of discrimination, or specificity, is best measured in experiments that compare the T.sub.m values of duplexes having perfect Watson/Crick complementarity versus those with one or more mismatches (e.g. A+G or A+C). The destabilization, seen by the decrease in T.sub.m, is a measure of specificity, pertinent to structural modifications, hybridization conditions, or other experimental parameters. Although some nucleic acid analogs have superior properties, most show impaired and deficient thermal melting values.
Additionally, some nucleic acids and analogs can form higher order structures than duplexes. For example, triplex structures involve three strands bound in a sequence dependent manner. While higher order structures exist in nature and play important roles in gene expression, recombination, and replication, they can lessen or complicate the intended, targeted activity of an exogenous nucleic acid analog. Therefore, it is desirable for most purposes that nucleic acid analogs have clear and predictable molecular recognition properties. The most desirable molecular recognition properties of a nucleic acid analog are high affinity and specificity in Watson/Crick base-pairing.
Exogenous nucleic acids outside the cell nucleus and replicative organelles are rapidly degraded and metabolized by enzymes. Structural analogs of nucleic acids often are poor substrates for phosphodiesterase, exo- and endonucleases which rapidly degrade foreign DNA and RNA. Thus, nuclease-resistant analogs attain a higher, more stable intra-cellular concentration and can exert their antisense, and other hybridization-dependent effects, over a useful period of time in vitro or in vivo. It is desirable that nucleic acid analogs be nuclease-resistant.
Although many nucleic acid analogs have some desirable properties, such analogs may have numerous other properties that render them unsuitable for common molecular biology techniques such as PCR or nucleic acid sequencing. For example, peptide nucleic acids--PNA (Nielsen, P. E. etal, Science (1991) 254:1497-1500) cannot function as primer extension templates or primers because they are not substrates for ligase, polymerase, or restriction enzymes. Accordingly, it is of interest to provide nucleic acid analogs that have such useful properties. It is also of interest to provide nucleic acid analogs that have one or more properties that are advantageous with respect to corresponding DNA molecules, but may also be used in a variety of molecular biology methods including annealing, ligation, sequencing, cleavage, PCR, and other primer extension reactions. It is of further interest to provide methods of using such analogs.